An HPLC method with ultraviolet-visible spectrophotometry detection has been optimized and validated for the simultaneous determination of phenolic compounds, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) as antioxidants, and octyl methyl cinnamate (OMC) as UVB-filter in several personal care products. The dynamic range was between 1 to 250 mg/L with relative standard deviation less than 0.25% (
Phenolic compounds such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) act as antioxidants and octyl methyl cinnamate (OMC) as UVB-filter are active compounds in personal care products (see Figure
Structures of common phenolic compounds in personal care products.
BHA and BHT are addedsingly or in combination to prevent oxidative rancidity in personal care products [
Reversed phase HPLC with UV/Vis detector (RP-HPLC-UV/Vis) is an important analytical technique with strong chromophores that absorb light in the wavelength region from 200 nm to 800 nm [
12 personal care samples were collected from local supermarket in Kuching city. Four types of personal care products were collected, that is, sunscreen cream, milk lotion, hair gel, and hair oil. The personal care samples were manufactured in Malaysia, Thailand, Indonesia, and Philippines.
All chemical reagents used for analysis phenolic compounds by RP-HPLC-UV/Vis were analytical Grade (99.99%) of Merck (Darmstadt, Germany). The reagents include n-hexane, methanol, ethanol, and acetonitrile. Reverse-osmosis type quality water was used during analysis. Standards of butylated hydroxyanisole BHA (96%), butylated hydroxytoluene BHT (99.8%), and OMC octyl methoxy cinnamate (98%) were purchased from Acros-Organics (New Jersey, USA).
An individual of 5000 mg/L stock solution of BHA, BHT, and OMC in acetonitrile was prepared by weighing equivalent accurately 1250 mg each of BHA, BHT, and OMC in the flask and diluted with 100 mL acetonitrile. The mixture was shaken until a homogenous and clear solution formed and added with acetonitrile until final volume of 250 mL. The stock solution was covered with aluminum foil and stored in a freezer (4°C) and away from light for a maximum of one month. Prior to analysis, standard working solutions were prepared by diluting appropriate amounts of the stock solutions in acetonitrile.
Extraction of BHA, BHT, and OMC from cosmetic samples was performed according to method described by Capitán-Vallvey et al. [
The quantitative and qualitative analysis of phenolic compounds was performed on Shimadzu HPLC system model LC-20AT equipped with four pumps and Shimadzu SPD-20 AV UV/Vis detector. 50
The UV spectrum of BHA, BHT, and OMC exhibited maximum absorption at 290, 275 and 300 nm, respectively. For the RP-HPLC analysis, the UV/Vis detector was fixed at 280 nm as maximum wave length (
pH is an important parameter to be optimized as it affects the ionization of phenolic compounds. Separation of BHA, BHT, and OMC are sensitive to the pH values because at low pH values, phenolic antioxidants are ionized due to the increase of protonation in mobile phase [
Effect of acetic acid percentage in phase B of mobile phase on pH, resolution factors, and total analysis time.
Acetic acid concentration (%, v/v) | 0 | 0.5 | 1 | 1.5 | 2 |
---|---|---|---|---|---|
pH value | 7 | 4 | 3.5 | 3.2 | 3 |
Resolution factors ( | 0.79 | 1.92 | 1.98 | 1.99 | 2 |
Total time of elute the analytes (minutes) | 8.5 | 6.0 | 5.5 | 5.3 | 5.3 |
Variation of resolution factor between BHT and OMC at different pH values of phase B of mobile phase.
It was observed that the resolution factor (
Chromatogram of BHA, BHT, and OMC analyzed using RP-HPLC-UV/Vis at
Flow rate of mobile phase has important effect on retention time, and peak area and little effect on separation for BHA, BHT, and OMC. Table
The retention times of BHA, BHT, and OMC at different flow rate of mobile phase.
Flow rate (mL/min) | Retention time of BHA (minutes) | Retention time of BHT (minutes) | Retention time of OMC (minutes) |
---|---|---|---|
0.10 | 21.18 | 34.93 | 40.69 |
0.15 | 13.98 | 22.81 | 26.48 |
0.20 | 10.53 | 16.89 | 19.49 |
0.25 | 8.59 | 14.49 | 16.99 |
0.30 | 7.02 | 11.22 | 12.94 |
0.35 | 5.90 | 9.09 | 10.44 |
0.40 | 5.34 | 8.86 | 9.93 |
0.45 | 4.97 | 8.08 | 8.92 |
0.50 | 4.3 | 6.74 | 7.74 |
0.55 | 3.82 | 6.05 | 6.95 |
0.60 | 3.49 | 5.51 | 6.33 |
0.65 | 3.21 | 5.03 | 5.79 |
0.70 | 3.03 | 5.03 | 5.85 |
0.75 | 2.82 | 4.60 | 5.33 |
0.80 | 2.65 | 4.35 | 5.05 |
0.85 | 2.35 | 3.79 | 4.37 |
0.90 | 2.33 | 3.72 | 4.29 |
0.95 | 2.22 | 3.63 | 4.19 |
1.00 | 2.09 | 3.29 | 3.79 |
1.05 | 1.97 | 3.06 | 3.62 |
1.10 | 1.92 | 3.05 | 3.58 |
1.15 | 1.87 | 3.01 | 3.56 |
1.20 | 1.81 | 2.94 | 3.48 |
1.25 | 1.72 | 2.85 | 3.29 |
Figure
Effect of mobile phase composition on retention time of BHA, BHT, and OMC.
The validation study for BHA, BHT, and OMC using RP-HPLC-UV/Vis was performed under the optimized conditions at 280 nm as maximum wave length, 0.8 mL/min as flow rate of mobile phase, and mixture phase A (acetonitrile) with phase B (water:acetic acid; 99 : 1; v/v) as mobile phase with elution ratio (90A : 10B; v/v) during the analysis time (8 minutes).
Eight standards solution of BHA, BHT, and OMC in acetonitrile concentrations of 1, 10, 25, 50, 75, 100, 125, and 250 mg/L were prepared. The calibration curves obtained by plotting the peak area of chromatograms for BHA, BHT, and OMC against the concentration are presented in Figure
Validation of analytical method for BHA, BHT, and OMC by RP-HPLC-UV/Vis.
Compound | Retention time (minutes) | Calibration equation | RSD% | LOD (mg/L) | LOQ (mg/L) | |
---|---|---|---|---|---|---|
BHA | 2.60 | 0.999 | 0.18 | 0.196 | 0.593 | |
BHT | 4.35 | 0.999 | 0.17 | 0.170 | 0.515 | |
OMC | 4.95 | 0.999 | 0.25 | 0.478 | 1.448 |
Calibration curves for BH, BHT, and OMC analysed on RP-HPLC-UV/Vis at
LOD for BHA and BHT by RP-HPLC-UV/Vis in this study (0.196 and 0.170 mg/L, resp.) are low compared with previous publications for LOD of BHA and BHT reported by Capitán-Vallvey et al. [
The relative recoveries for phenolic compounds were determined by using the external standard additions methodology at four spiked levels 1, 5, 10, and 25 mg/L by comparison with a standard chromatogram of similar concentration. Mean recoveries for every spiked level were determined at three times with four replicates representing at each time (see Table
Results of recovery study for BHA, BHT, and OMC by RP-HPLC-UV/Vis at
Relative recovery (%, | ||||||
Spiked (mg/L) | BHA | RSD% | BHT | RSD% | OMC | RSD% |
1 | 105.9 | 2.64 | 108.9 | 7.69 | 103.7 | 2.53 |
5 | 102.3 | 3.72 | 102.8 | 4.02 | 94.6 | 1.95 |
10 | 99.7 | 1.65 | 95.9 | 3.13 | 93.3 | 1.45 |
25 | 92.1 | 1.18 | 83.2 | 2.24 | 87.3 | 1.27 |
The recovery ranges of BHA and BHT in this study (92.1%–105.9%, 83.2%–108.9%, resp.) are better than previous paper by Saad et al. [
Four types of personal care products such as sunscreen cream, milk lotion, hair gel and hair oil with three different samples for every type were analyzed for their BHA, BHT, and OMC content as can be seen in Table
Concentration of BHA, BHT, and OMC in sunscreen cream, milk lotion, hair gel, and hair oil samples determined by RP-HPLC-UV/Vis at
Type | Commercial name | Country of origin | Phenolic compounds | Mean concentration (mg/g) | ||||
(1) ( | (2) ( | (3) ( | Average (mg/g) | RSD% | ||||
Sunscreen cream | Aiken | Malaysia | BHA | 4.85 | 1.50 | |||
BHT | 1.33 | 3.88 | ||||||
OMC | 65.5 | 0.77 | ||||||
Nivea | Thailand | BHA | 3.26 | 2.43 | ||||
BHT | 1.01 | 4.47 | ||||||
OMC | 27.96 | 1.58 | ||||||
Gervenne | Malaysia | BHA | 1.82 | 3.92 | ||||
BHT | n.d | n.d | n.d | n.d | n.d | |||
OMC | 16.23 | 2.68 | ||||||
Milk lotion | Nivea | Thailand | BHA | 4.50 | 1.57 | |||
BHT | 2.30 | 3.21 | ||||||
OMC | 13.83 | 1.55 | ||||||
New Trendy | Malaysia | BHA | 4.16 | 2.82 | ||||
BHT | n.d | n.d | n.d | n.d | n.d | |||
OMC | 8.99 | 3.79 | ||||||
Garnier | Indonesia | BHA | 2.74 | 3.32 | ||||
BHT | 0.73 | 3.26 | ||||||
OMC | 17.0 | 1.86 | ||||||
Hair gel | De Boy | Malaysia | BHA | 1.28 | 3.14 | |||
BHT | 0.22 | 3.40 | ||||||
OMC | 0.13 | 4.52 | ||||||
Beyond | Malaysia | BHA | 1.38 | 3.37 | ||||
BHT | 0.16 | 4.05 | ||||||
OMC | 0.30 | 3.48 | ||||||
Elite | Malaysia | BHA | 1.51 | 2.76 | ||||
BHT | 0.17 | 4.48 | ||||||
OMC | 0.84 | 2.69 | ||||||
Hair oil | Elite | Malaysia | BHA | 3.89 | 1.06 | |||
BHT | 0.87 | 2.11 | ||||||
OMC | 0.82 | 1.37 | ||||||
Gervenne | Malaysia | BHA | 0.13 | 4.66 | ||||
BHT | 1.54 | 3.25 | ||||||
OMC | 3.40 | 1.75 | ||||||
Johnsons | Philippines | BHA | 0.30 | 3.40 | ||||
BHT | 0.18 | 4.13 | ||||||
OMC | 0.57 | 2.19 |
n.d: not detected or below detection limit.
Table
Chromatogram of BHA, BHT, and OMC in Aiken sunscreen cream sample using RP-HPLC-UV/Vis at
Chromatogram of BHA, BHT, and OMC in Nivea milk lotion sample using RP-HPLC-UV/Vis at
Table
Table
Chromatogram of BHA, BHT, and OMC in De Boy hair gel sample using RP-HPLC-UV/Vis at
Chromatogram of BHA, BHT, and OMC in Elite hair oil sample using RP-HPLC-UV/Vis at
Table
The analytical method by RP-HPLC-UV/Vis in this study is modern for simultaneous determination of common phenolic compounds in personal care products. The optimum parameters that can be used are as follows; binary mixture of phase A (acetonitrile) and phase B (water/acetic acid, 99 : 1, v/v) as mobile phase with elution ratio (90 A: 10 B, v/v) during the analysis time (8 minutes), pH 3.5 of phase B (using acetic acid for adjust it), 0.8 mL/min as flow rate and 280 nm as maximum wave length. The satisfactory results of optimization and validation methods are quick, accurate, sensitive, excellent recoveries, convenient and effective for phenolic compounds. The developed method was successfully applied to fingerprint analysis of personal care products as well as quantify the relevant phenolic compounds markers present in these products under optimum parameters. This method can be applied to analyze the phenolic compounds in commercial cosmetic and food products.
The authors would like to acknowledge Department of Chemistry, Faculty of Resource science and Technology, UNIMAS, for providing HPLC instrument for this study.